Parenteral use of bacterial phage associated lysing enzymes for the therapeutic treatment of bacterial infections

ABSTRACT

The present invention discloses a method and composition for the treatment of bacterial infections by the parenteral introduction of at least one lytic enzyme produced by a bacteria infected with a bacteriophage specific for that bacteria and an appropriate carrier for delivering the lytic enzyme into a patient. The injection can be done intramuscularly, subcutaneously, or intravenously.

The following application is a continuation in part of U.S. patentapplication Ser. No. 09/395,636, filed Sep. 14, 1999, now U.S. Pat. No.6,056,954, issued May 2, 2000, which is a continuation in part of U.S.patent application Ser. No. 08/962,523, filed Oct. 31, 1997, now U.S.Pat. No. 5,997,862, issued Dec. 7, 1999.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention discloses a method and composition for thetreatment of bacterial infections by the parenteral introduction of alysing enzyme blended with an appropriate carrier into a patient. Theinjection can be done intramuscularly, subcutaneously, or intravenously.

2. Description of the Prior Art

In the past, antibiotics have been used to treat various infections. Thework of Selman Waksman in the introduction and production ofStreptomycetes, and Dr. Fleming's discovery of penicillin, as well asthe work of numerous others in the field of antibiotics, are well known.Over the years, there have been additions and chemical modifications tothe “basic” antibiotics in attempts to make them more powerful, or totreat people allergic to these antibiotics.

Others have found new uses for these antibiotics. U.S. Pat. No.5,260,292 (Robinson et al.) discloses the topical treatment of acne withaminopenicillins. The method and composition for topically treating acneand acneiform dermal disorders includes applying an amount of anantibiotic, effective for treating acne and acneiform dermal disorders,selected from the group consisting of ampicillin, amoxicillin, otheraminopenicillins, and cephalosporins, and derivatives and analogsthereof. U.S. Pat. No. 5,409,917 (Robinson et al.) discloses the topicaltreatment of acne with cephalosporins.

However, as more antibiotics have been prescribed or used at an everincreasing rate for a variety of illnesses, increasing numbers ofbacteria have developed a resistance to antibiotics. Larger doses ofstronger antibiotics are now being used to treat ever more resistantstrains of bacteria. Consequently, multiple antibiotic resistantbacteria have been developed. The use of more antibiotics and the numberof bacteria showing resistance has led to increases in the amount oftime that the antibiotics need to be used. Broad, non-specificantibiotics, some of which have detrimental effects on the patient, arenow being used more frequently. Additionally, the number of peopleshowing allergic reactions to antibiotics appears to be increasing.

Consequently, other efforts have been sought to first identify and thenkill bacteria.

Attempts have been made to treat bacterial diseases with by the use ofbacteriophages. U.S. Pat. No. 5,688,501 (Merril, et al.) discloses amethod for treating an infectious bacterial disease with lytic ornon-lytic bacteriophages that are specific for particular bacteria.

U.S. Pat. No. 4,957,686 (Norris) discloses a procedure of improveddental hygiene which comprises introducing into the mouth bacteriophagesparasitic to bacteria which possess the property of readily adhering tothe salivary pellicle.

It is to be noted that the direct introduction of bacteriophages into ananimal to prevent or fight diseases has certain drawbacks. Specifically,the bacteria must be in the right growth phase for the phage to attach.Both the bacteria and the phage have to be in the correct andsynchronized growth cycles. Additionally, there must be the right numberof phages to attach to the bacteria; if there are too many or too fewphages, there will either be no attachment or no production of thelysing enzyme. The phage must also be active enough. The phages are alsoinhibited by many things including bacterial debris from the organismthe phages are going to attack. Further complicating the direct use ofbacteriophage is the possibility of immunological reactions, renderingthe phage non-functional.

Consequently, others have explored the use of other safer and moreeffective means to treat and prevent bacterial infections.

One bacteria for which a more effective treatment has been extensivelyexplored is Streptococcus. The genus Streptococcus is comprised of awide variety of both pathogenic and commensal gram-positive bacteriawhich are found to inhabit a wide range of hosts, including humans,horses, pigs, and cows. Within the host, streptococci are often found tocolonize the mucosa surfaces of the mouth, nares and pharynx. However,in certain circumstances, they may also inhabit the skin, heart ormuscle tissue.

Pathogenic streptococci of man include S. pyogenes, S. pneumoniae, andS. faecalis. While Group A streptococci may be present in the throat oron the skin and cause no symptoms of disease, they may also causeinfections that range from mild to severe, and even life-threatening.Among the pathogenic hemolytic streptococci, S. pyogenes, or group Astreptococci have been implicated as the etiologic agent of acutepharyngitis (“strep throat”), impetigo, rheumatic fever, scarlet fever,glomerulonephritis, and invasive fasciitis. Necrotizing fasciitis(sometimes described by the media as “the flesh-eating bacteria”) is adestructive infection of muscle and fat tissue. Invasive group Astreptococcal infections occur when the bacteria get past the defensesof the person who is infected. About 10,000-15,000 cases of invasive GASdisease occur in the United States each year, resulting in over 2,000deaths. CDC estimates that 500 to 1,500 cases of necrotizing fasciitisand 2,000 to 3,000 cases of streptococcal toxic shock syndrome occureach year in the United States. Approximately 20% of patients withnecrotizing fasciitis die, and 60% of patients with streptococcal toxicshock syndrome die. About 10 to 15% of patients with other forms ofinvasive group A streptococcal disease die.

Additionally, Group C Streptococcus can cause cellulitis from skinbreaks, although cellulitis is normally associated with Staphylococcusaureus. Cellulitis can result in death, particularly in olderindividuals or in individuals who are already weakened.

Reports have described the characteristics of an enzyme produced by thegroup C streptococcal organism after being infected with a particularbacteriophage identified as Cl (Maxted, W. R. “The Active Agent inNascent Page Lywsis of Streptococci,” J. Gen Micro., vol 16, pp585-595,1957, Krause, R. M., “Studies on the Bacteriophages of HemolyticStreptococci,” J. Exp. Med, vol. 108, pp 803-821, 1958) and Fischetti,(Fischetti, V. A., et al, “Purification and Physical Properties of GroupC Streptococcal Phage Associated Lysin,” J. Exp. Med, Vol 133 pp.1105-1117, 1971). The enzyme was given the name lysin and was found tospecifically cleave the cell wall of group A, group C, and group Estreptococci. These investigators provided information on thecharacteristics and activities of this enzyme with regard to lysing thegroup A streptococci and releasing the cell wall carbohydrate.

U.S. Patent Application Ser. No. 08/962,523) (Fischetti, et. al.) andU.S. Patent Application Ser. No. 09/257,026) (Fischetti et al.) disclosethe use of an oral delivery mode, such as a candy, chewing gum, lozenge,troche, tablet, a powder, an aerosol, a liquid or a liquid spray,containing a lysin enzyme produced by group C streptococcal bacteriainfected with a Cl bacteriophage for the prophylactic and therapeutictreatment of Streptococcal A throat infections, commonly known as strepthroat.

U.S. Patent Application No. 6,056,954 (Fischetti, et. al) discloses amethod for the prophylactic and therapeutic treatment of bacterialinfections which comprises the treatment of an individual with aneffective amount of a lytic enzyme composition specific for theinfecting bacteria, and a carrier for delivering said lytic enzyme. Themethods disclosed included the topical, oral, and respiratory methods ofdelivering the enzyme. Another method disclosed in that applicationincludes the use of suppositories. These methods and compositions can beused for the treatment of upper respiratory infections, skin infections,wounds, and burns, vaginal infections, eye infections, intestinaldisorders and dental problems.

U.S. Patent Application No. 6,056,955 (Fischetti et al.) discloses amethod and composition for the topical treatment of streptococcalinfections by the use of a lysin enzyme blended with a carrier suitablefor topical application to dermal tissues. The method for the treatmentof dermatological streptococcal infections comprises administering acomposition comprising an effective amount of a therapeutic agent, withthe therapeutic agent comprising a lysin enzyme produced by group Cstreptococcal bacteria infected with a Cl bacteriophage. The therapeuticagent can be in a pharmaceutically acceptable carrier.

The use of phage associated lytic enzymes produced by the infection of abacteria with a bacteria specific phage has numerous advantages for thetreatment of diseases. As the phage are targeted for specific bacteria,the lytic enzymes do not interfere with normal flora. Also, lytic phagesprimarily attack cell wall structures which are not affected by plasmidvariation. The actions of the lytic enzymes are fast and do not dependon bacterial growth.

However, sometimes the bacterial infections, by the time they aretreated, have developed into more serious illnesses. For example,dermatological infections such as Staphylococcus aureus andStreptococcal pneumoniae can develop into cellulitis, which, unchecked,can lead to a degradation of the connective tissue, septicemia, andpossibly death. Other bacterial infections can also evolve into deeptissue infections. Other infections by other bacteria, not necessarilydermatological by nature, can infect and localize in certain tissues ofthe body, making the infections difficult to treat.

SUMMARY OF THE INVENTION

The present invention discloses the use of a variety of bacterial phageassociated lytic enzymes for the treatment of a wide variety ofillnesses caused by bacterial infections. More specifically, the presentinvention discloses the PARENTERAL application of a bacterial lyticenzyme, wherein the phage associated lytic enzyme is administeredintramuscularly, subdermally, subcutaneously, or intravenously to treata bacterial infection.

It is another object of the invention to apply a phage associated lyticenzyme intravenously, to treat septicemia and general infections.

It is also an object of the invention to inject a phage associated lyticenzyme into the tissue of an organism to treat a deep tissue infection.

It is also an object of the invention to administer a phage associatedlytic enzyme intravenously.

The invention (which incorporates U.S. Pat. No. 5,604,109 in itsentirety by reference) uses an enzyme produced by the bacterial organismafter being infected with a particular bacteriophage as a therapeutictreatment for those who have already become ill from the infection. Thepresent invention is based upon the discovery that phage lytic enzymesspecific for bacteria infected with a specific phage can effectively andefficiently break down the cell wall of the bacterium in question. Atthe same time, the semipurified enzyme is lacking in proteolyticenzymatic activity and therefore non-destructive to mammalian proteinsand tissues when present during the digestion of the bacterial cellwall.

In one embodiment of the invention, the treatments of a variety ofillnesses caused by Streptococcus fasciae, and Staphylococcus aureus aredisclosed.

In yet another embodiment of the invention, lysostaphin, the enzymewhich lyses Staphylococcus aureus, can be included in the therapeuticagent.

In a further embodiment of the invention, conventional antibiotics maybe included in the therapeutic agent with the lytic enzyme, and with orwithout the presence of lysostaphin.

In another embodiment of the invention, more than one lytic enzyme mayalso be included in the therapeutic agent.

The therapeutic agent may be given parenterally, by means of anintramuscular, intradermal, or subcutaneous injection, or the agent maybe given intravenously.

DETAILED DESCRIPTION OF THE INVENTION

The method for treating systemic or tissue bacterial infectionscomprises parenterally treating the infection with a therapeutic agentcomprising an effective amount of at least one lytic enzyme produced bya bacteria infected with a bacteriophage specific for the bacteria, andan appropriate carrier.

The composition may be used for the therapeutic treatment ofPseudomonas, Clostridium, Staphylococcus infections, among others. Theseand other bacteria can be infected with bacteriophage specific for saidbacteria, whereupon a lytic enzyme is produced specific for the lysingof that bacteria. For example, the composition which may be used for thetherapeutic treatment of a strep infection includes the lysin enzyme anda means of application. When group C Streptococci are infected with a Clbacteriophage, a lysin enzyme is produced specific for the lysing ofStreptococcus group A.

A number of different bacteria may be treated. Among the bacteria whichmost often infect deep tissues, and, more specifically connectivetissues, are Group A Streptococcus, Staphylococcus, Pseudomonas, andClostridium. More than one lytic enzyme may be introduced into theinfected body at a time.

A number of different methods may be used to introduce the lyticenzyme(s). These methods include introducing the lytic enzymeintravenously, intramuscularly, subcutaneously, and subdermally.

In one preferred embodiment of the invention, a deep tissue infectionmay be treated by injecting into the infected tissue of the patient atherapeutic agent comprising the appropriate lytic enzyme(s) and acarrier for the enzyme. The carrier may be comprised of distilled water,a saline solution, albumin, a serum or any combinations thereof Morespecifically, solutions for infusion or injection may be prepared in aconventional manner, e.g. with the addition of preservatives such asp-hydroxybenzoates or stabilizers such as alkali metal salts ofethylene-diamine tetraacetic acid, which may then be transferred intofusion vessels, injection vials or ampules. Alternatively, the compoundfor injection may be lyophilized either with or without the otheringredients and be solubilized in a buffered solution or distilledwater, as appropriate, at the time of use. Non-aqueous vehicles such asfixed oils and ethyl oleate are also useful herein.

In cases where intramuscular injection is the chosen mode ofadministration, an isotonic formulation is preferably used. Generally,additives for isotonicity can include sodium chloride, dextrose,mannitol, sorbitol and lactose. In some cases, isotonic solutions suchas phosphate buffered saline are preferred. Stabilizers include gelatinand albumin. In some embodiments, a vasoconstriction agent is added tothe formulation. The pharmaceutical preparations according to thepresent invention are provided sterile and pyrogen free.

The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; glycine; amino acids such as glutamic acid,aspartic acid, histidine, or arginine; monosaccharides, disaccharides,and other carbohydrates including cellulose or its derivatives, glucose,mannose, trehalose, or dextrins; chelating agents such as EDTA, sugaralcohols such as mannitol or sorbitol; counter-ions such as sodium;non-ionic surfactants such as polysorbates, poloxamers,orpolyethyleneglycol (PEG); and/orneutral salts, e.g., NaCl, KCl,MgCl.sub.2, CaCl.sub.2, etc.

Glycerin or glycerol (1,2,3-propanetriol) is commercially available forpharmaceutical use. It may be diluted in sterile water for injection, orsodium chloride injection, or other pharmaceutically acceptable aqueousinjection fluid, and used in concentrations of 0.1 to 100% (v/v),preferably 1.0 to 50% more preferably about 20%.

DMSO, which is an aprotic solvent with a remarkable ability to enhancepenetration of many locally applied drugs. DMSO may be diluted insterile water for injection, or sodium chloride injection, or otherpharmaceutically acceptable aqueous injection fluid, and used inconcentrations of 0.1 to 100% (v/v).

The carrier vehicle may also include Ringer's solution, a bufferedsolution, and dextrose solution, particularly when an intravenoussolution is prepared.

Prior to, or at the time the lysin enzyme is put in the carrier systemor oral delivery mode, it is preferred that the enzyme be in astabilizing buffer environment for maintaining a pH range between about4.0 and about 9.0, more preferably between about 5.5 and about 7.5 andmost preferably at about 6.1. This is pH range is most suitable for thelysin enzyme for Streptococcus.

The stabilizing buffer should allow for the optimum activity of thelysin enzyme. The buffer may be a reducing reagent, such asdithiothreitol. The stabilizing buffer may also be or include a metalchelating reagent, such as ethylenediaminetetracetic acid disodium salt,or it may also contain a phosphate or citrate-phosphate buffer. Thebuffers found in the carrier can serve to stabilize the environment forthe lytic enzymes.

The effective dosage rates or amounts of the lytic enzyme to treat theinfection, and the duration of treatment will depend in part on theseriousness of the infection, the duration of exposure of the recipientto the infectious bacteria, the number of square centimeters of skin ortissue which are infected, the depth of the infection, the seriousnessof the infection, and a variety of a number of other variables. Thecomposition may be applied anywhere from once to several times a day,and may be applied for a short or long term period. The usage may lastfor days or weeks. Any dosage form employed should provide for a minimumnumber of units for a minimum amount of time. The concentration of theactive units of enzyme believed to provide for an effective amount ordosage of enzyme may be in the range of about 100 units/ml to about500,000 units/ml of composition, preferably in the range of about 1000units/ml to about 100,000 units/ml, and most preferably from about10,000 to 100,000 units/ml. The amount of active units per ml and theduration of time of exposure depends on the nature of infection, and theamount of contact the carrier allows the lytic enzyme to have. It is tobe remembered that the enzyme works best when in a fluid environment.Hence, effectiveness of the enzyme is in part related to the amount ofmoisture trapped by the carrier. For the treatment of septicemia, thereshould be a continuous intravenous flow of therapeutic agent into theblood stream. The concentration of lytic enzyme for the treatment ofsepticemia is dependent upon the seriousness of the infection.

In order to accelerate treatment of the infection, the therapeutic agentmay further include at least one complementary agent which can alsopotentiate the bactericidal activity of the lytic enzyme. Thecomplementary agent can be penicillin, synthetic penicillins bacitracin,methicillin, cephalosporin, polymyxin, cefaclor. Cefadroxil,cefamandolenafate, cefazolin, cefixime, cefinetazole, cefonioid,cefoperazone, ceforanide, cefotanme, cefotaxime, cefotetan, cefoxitin,cefpodoxime proxetil, ceftazidime, ceftizoxime, ceftriaxone, cefriaxonemoxalactam , cefuroxime, cephalexin, cephalosporin C, cephalosporin Csodium salt, cephalothin, cephalothin sodium salt, cephapirin,cephradine, cefuroximneaxetil, dihydratecephalothin, moxalactam,loracarbef, mafate, chelating agents and any combinations thereof inamounts which are effective to synergistically enhance the therapeuticeffect of the lytic enzyme.

Additionally, the therapeutic agent may further comprise the enzymelysostaphin for the treatment of any Staphylococcus aureus bacteria.Mucolytic peptides, such as lysostaphin, have been suggested to beefficacious in the treatment of S. aureus infections of humans(Schaffner et al., Yale J. Biol. & Med., 39:230 (1967) and bovinemastitis caused by S. aureus (Sears et al., J. Dairy Science, 71 (Suppl.1): 244(1988)). Lysostaphin, a gene product of Staphylococcus simulans,exerts a bacteriostatic and bactericidal effect upon S. aureus byenzymatically degrading the polyglycine cross-link of the cell wall(Browder et al., Res. Comm., 19: 393-400 (1965)). U.S. Pat. No.3,278,378 describes fermentation methods for producing lysostaphin fromculture media of S. staphylolyticus, later renamed S. simulans. Othermethods for producing lysostaphin are further described in U.S. Pat.Nos. 3,398,056 and 3,594,284. The gene for lysostaphin has subsequentlybeen cloned and sequenced (Recsei et al., Proc. Natl. Acad. Sci. USA,84: 1127-1131 (1987)). The recombinant mucolytic bactericidal protein,such as r-lysostaphin, can potentially circumvent problems associatedwith current antibiotic therapy because of its targeted specificity, lowtoxicity and possible reduction of biologically active residues.Furthermore, lysostaphin is also active against non-dividing cells,while most antibiotics require actively dividing cells to mediate theireffects (Dixon et al., Yale J. Biology and Medicine, 41: 62-68 (1968)).Lysostaphin, in combination with the lysin enzyme, can be used in thepresence or absence of the listed antibiotics. There is a degree ofadded importance in using both lysostaphin and the lysin enzyme in thesame therapeutic agent. Frequently, when a body has a bacterialinfection, the infection by one genus of bacteria weakens the body orchanges the bacterial flora of the body, allowing other potentiallypathogenic bacteria to infect the body. One of the bacteria thatsometimes co-infects a body is Staphylococcus aureus. Many strains ofStaphylococcus aureus produce penicillinase, such that Staphylococcus,Streptococcus, and other gram positive bacterial strains will not bekilled by standard antibiotics. Consequently, the use of the lysin andlysostaphin, possibly in combination with antibiotics, can serve as themost rapid and effective treatment of bacterial infections. In yetanother preferred embodiment, the invention may include mutanolysin, andlysozyme.

Many modifications and variations of the present invention are possiblein light of the above teachings. It is, therefore, to be understoodwithin the scope of the appended claims the invention may be protectedotherwise than as specifically described.

What we claim is:
 1. A method for the treatment of bacterial infections,comprising: administering parentally in a host a composition comprisingan effective amount of at least one lytic enzyme produced by a bacteriainfected with a bacteriophage specific for said bacteria and a carrierfor delivering said lytic enzyme to the to the site of the infection. 2.The method according to claim 1, wherein the at least one lytic enzymeis for the treatment of Pseudomonas.
 3. The method according to claim 1,wherein the at least one lytic enzyme is for the treatment ofStreptococcus.
 4. The method according to claim 1, wherein the at leastone lytic enzyme is for the treatment of Staphylococcus.
 5. The methodaccording to claim 1, wherein the at least one lytic enzyme is for thetreatment of Clostridium.
 6. The method according to claim 1, whereinsaid composition further comprises a buffer that maintains pH of thecomposition at a range between about 4.0 and about 9.0.
 7. The methodaccording to claim 6, wherein the buffer maintains the pH of thecomposition at the range between about 5.5 and about 7.5.
 8. The methodaccording to claim 6, wherein said buffer comprises a reducing reagent.9. The method according to claim 8, wherein said reducing reagent isdithiothreitol.
 10. The method according to claim 6, wherein said buffercomprises a metal chelating reagent.
 11. The method according to claim10, wherein said metal chelating reagent is ethylenediaminetetraceticdisodium salt.
 12. The method according to claim 6, wherein said bufferis a citrate-phosphate buffer.
 13. The method according to claim 1,further comprising a bactericidal or bacteriostatic agent as apreservative.
 14. The method according to claim 1, wherein said at leastone lytic enzyme is lyophilized.
 15. The method according to claim 1,wherein said therapeutic agent is administered intravenously.
 16. Themethod according to claim 1, wherein said therapeutic agent isadministered intramuscularly.
 17. The method according to claim 1,wherein said therapeutic agent is administered subcutaneously.
 18. Themethod according to claim 1, wherein the therapeutic agent furthercomprises at least one complementary agent which potentiates thebactericidal activity of the lysine enzyme, said complementary agentbeing selected from the group consisting of penicillin, syntheticpenicillins, bacitracin, methicillin, cephalosporin, polymyxin,cefaclor, Cefadroxil, cefamandole nafate, cefazolin, cefixime,cefinetazole, cefonioid, cefoperazone, ceforanide, cefotanme,cefotaxime, cefotetan, cefoxitin, cefpodoxime proxetil, ceftazidime,ceftizoxime, ceftriaxone, cefriaxone moxalactam, cefuroxime, cephalexin,cephalosporin C, cephalosporin C sodium salt, cephalothin, cephalothinsodium salt, cephapirin, cephradine, cefuroximneaxetil,dihydratecephalothin, moxalactam, and chelating agents in an amounteffective to synergistically enhance the therapeutic effect of the lysinenzyme.
 19. The method according to claim 1, wherein said carriercomprises of distilled water, a saline solution, albumin, a serum, andany combinations thereof.
 20. The method according to claim 1, whereinsaid carrier further comprises preservatives.
 21. The method accordingto claim 20, wherein said preservatives comprise p-hydroxybenzoates. 22.The method according to claim 1, wherein said carrier comprises anisotonic solution for an injection, said isotonic solution comprising acompound selected from group consisting of sodium chloride, dextrose,mannitol, sorbitol, lactose, phosphate buffered saline, gelatin,albumin, a vasoconstriction agent and combinations.
 23. The methodaccording to claim 22, wherein said further carrier further comprisesDMSO.